Download Catecholaminergic neurons in rat dorsal motor nucleus of vagus

Am J Physiol Gastrointest Liver Physiol
280: G361–G367, 2001.
Catecholaminergic neurons in rat dorsal motor nucleus
of vagus project selectively to gastric corpus
JINFENG J. GUO,1 KIRSTEEN N. BROWNING,1
RICHARD C. ROGERS,2 AND R. ALBERTO TRAVAGLI1
1
Neurogastroenterology Research, Henry Ford Health System, Detroit, Michigan 48202;
and 2Department of Neuroscience, Ohio State University, Columbus, Ohio 43210
Received 28 June 2000; accepted in final form 25 September 2000
GASTRIC RELAXATION REFERS to the behavior of the stomach during food ingestion and is characterized by a
pronounced capacity to receive large increases in volume with only a slight increase in gastric pressure (1).
The vagus nerve provides an essential role in the
mediation of this relaxation reflex via an increase in
the nonadrenergic noncholinergic (NANC) inhibitory
innervation as well as a decrease of cholinergic excitatory innervation to the stomach (1, 20, 29, 34).
The dorsal motor nucleus of the vagus (DMV) supplies
parasympathetic motor preganglionic fibers to the viscera and is involved in the central nervous system (CNS)
control of gastric motility (10). Recently, we (29) have
provided physiological evidence that at least two types of
vagal motoneurons are involved in the gastric relaxation
that follows esophageal distension.
Several studies (2, 3, 15, 17–19, 21, 22, 28, 35, 40, 43)
have shown that the DMV contains neurons with diverse neurochemical phenotypes. These discrete neurochemical subpopulations could potentially provide
differential CNS modulation of specific vago-vagal reflexes involved in, for example, gastric relaxation.
It is well established that the DMV in general and its
caudal portion in particular are composed of neurons that
show tyrosine hydroxylase immunoreactivity (TH-IR) (2,
13, 22, 28, 35, 37). The pattern of distribution of dopamine-␤-hydroxylase (D␤H) activity overlaps the distribution of TH-IR in the caudal dorsal brain stem (6, 13, 28).
Recently, Willing and Berthoud (37) reported that the
population of D␤H and TH-IR neurons in the dorsal vagal
complex (i.e., DMV and nucleus of the solitary tract, NTS)
were almost identical in number and distribution, arguing that the TH-IR positive neurons in the DMV are
capable of synthesizing norepinephrine. These TH-IR
positive caudal DMV neurons have been reported (2, 22)
to display choline acetyltransferase activity, making
them likely candidates for vagal motoneurons that
project to peripheral targets.
Studies (4, 19) have also focused on the presence and
role of nitric oxide (NO) in the brain stem vagal nuclei.
Anatomic and physiological evidence points to a possible role of the NO synthase (NOS)-IR positive DMV
neurons in vagally mediated gastric relaxation. We
(43) have shown recently that a subpopulation of DMV
neurons containing NOS-IR projects selectively to the
gastric fundus.
Immunocytochemical detection of the immediateearly genes encoding for the c-Fos protein allows detection of those neurons activated after a wide variety
of stimuli. Increased c-Fos expression in response to
administration of the gastric relaxant CCK or after
gastric distension has been reported (27, 37) in neurons
located in areas of the caudal dorsal brain stem that
include the DMV and NTS.
The goals of this study were threefold. We aimed to
1) investigate whether the TH-IR positive DMV neu-
Address for reprint requests and other correspondence: R. A.
Travagli, Univ. of Michigan, Division of Gastroenterology, 3912
Taubman Center, Ann Arbor, MI 48109-0362 (E-mail: travagli7
@hotmail.com).
The costs of publication of this article were defrayed in part by the
payment of page charges. The article must therefore be hereby
marked ‘‘advertisement’’ in accordance with 18 U.S.C. Section 1734
solely to indicate this fact.
stomach; esophagus; receptive relaxation; gastric motility
http://www.ajpgi.org
0193-1857/01 $5.00 Copyright © 2001 the American Physiological Society
G361
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Guo, Jinfeng J., Kirsteen N. Browning, Richard C.
Rogers, and R. Alberto Travagli. Catecholaminergic neurons in rat dorsal motor nucleus of vagus project selectively
to gastric corpus. Am J Physiol Gastrointest Liver Physiol
280: G361–G367, 2001.—Nitric oxide synthase-immunoreactive (NOS-IR) neurons in the rat caudal dorsal motor nucleus
of the vagus (DMV) project selectively to the gastric fundus
and may be involved in vagal reflexes controlling gastric
distension. This study aimed to identify the gastric projections of tyrosine hydroxylase-immunoreactive (TH-IR) DMV
neurons, whether such neurons colocalize NOS-IR, and if
they are activated after esophageal distension. Gastric-projecting neurons were identified after injection of retrograde
tracers into the muscle wall of the gastric fundus, corpus, or
antrum/pylorus before removal and processing of the brain
stems for TH- and NOS-IR. A significantly higher proportion
of corpus- compared with fundus- and antrum/pylorus-projecting neurons were TH-IR (14% compared with 4% and 2%,
respectively, P ⬍ 0.05). Colocalization of NOS- and TH-IR
was never observed in gastric-projecting neurons. In rats
tested for c-Fos activation after intermittent esophageal balloon distension, no colocalization with TH-IR was observed in
DMV neurons. These findings suggest that TH-IR neurons in
the caudal DMV project mainly to the gastric corpus, constitute a subpopulation distinct from that of nitrergic vagal
neurons, and are not activated on esophageal distension.
G362
TH-IR IN RAT DMV
rons project selectively to specific gastric areas; 2)
explore whether TH- and NOS-IR positive neurons are
colocalized within the same DMV neurons; and 3) examine whether TH-IR positive neurons are activated
on esophageal distension.
EXPERIMENTAL PROCEDURES
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A total of 47 Sprague-Dawley rats (25–35 days old) of either
sex were used. Animals were subdivided into the following six
groups. In group 1 (n ⫽ 6) or control, no injections were performed on the gastric walls. In group 2 (n ⫽ 8), tetramethylrhodamine isothiocyanate (TRITC)-filled latex microspheres were
injected into the wall of the gastric fundus. In group 3 (n ⫽ 10),
TRITC-filled latex microspheres were injected into the wall of
the gastric corpus. In group 4 (n ⫽ 8), TRITC-filled latex
microspheres were injected into the wall of the antral/pyloric
area (see below). In group 5 (n ⫽ 10), double immunocytochemistry (TH and NOS) was carried out, and no injections were
performed on the gastric walls. In group 6 (n⫽ 5), esophageal
distension followed by double immunocytochemistry (TH and
c-Fos) took place, and no injections were performed on the
gastric walls. Unless otherwise specified, all rats were injected
with fluorogold (Fluorochrome, Englewood, CO; 20 ␮g/1 ml
saline/rat, ip) to label vagal preganglionic neurons innervating
the subdiaphragmatic viscera allowing delineation of the
boundaries of the DMV (24, 43).
Using a custom-made anesthetic chamber, rats were anesthetized deeply (abolition of foot pinch withdrawal reflex) with
2-bromo-2-chloro-1,1,1-trifluoroethane (halothane), and the abdominal and thoracic areas were shaved and cleaned with 70%
ethanol. After an abdominal laparotomy, the stomach was freed
from the liver and reflected gently to one side to facilitate access
to the gastric wall. With a 5-␮l Hamilton syringe, rats in groups
2, 3, and 4 were injected with TRITC-filled latex microspheres
(1:4 vol/vol dilution with saline; 0.5–1 ␮l per injection, 5–10
sites per area) in the muscular and mucosal layers of the
stomach (fundus, corpus, or antrum/pylorus, respectively). The
syringe needle was inserted 2–3 mm into the stomach wall at
an angle of 2–5° to facilitate the spread of beads within the
stomach wall and to reduce the possibility of perforating
the mucosa, perceived as a drop in the resistance opposing the
needle penetration into the stomach wall. The laparotomy was
then closed with 5-0 silk sutures, and the rats were allowed to
recover for 5–15 days, permitting retrograde transport of the
fluorescent marker as described previously (43).
Rats in group 6 were anesthetized with pentobarbital
sodium (Nembutal; 75mg/kg, ip). The thoracic esophagus was
then cannulated with a distension balloon inserted to the
point where its tip meets resistance at the lower esophageal
sphincter. Balloon catheters were constructed as previously
described (29). The balloon was distended intermittently
(final intramural pressure, 14–18 mmHg; 1 s on and 4 s off)
for 60–90 min. At the end of the experiment, rats were
perfused transcardially with saline, followed by 4% paraformaldehyde in PBS solution (see below).
After extraction, brain stems from all groups were fixed in
Zamboni’s fixative overnight, rinsed three to five times with
a solution of Triton X-100 in PBS (PBS-TX; for composition,
see Composition of solutions), and stored in PBS-TX overnight at 4°C. The brain stem was then immersed in PBS
containing 2.5% sucrose before cutting coronal sections of 50
␮m thickness using a cryostat. The brain stem slices were
mounted on gelatin-coated coverslips and preincubated in a
humid chamber with PBS-TX-BSA at 37°C for 30 min.
The slices from rats in groups 1–4 were rinsed with fresh
PBS-TX-BSA solution and incubated at 37°C for 2 h with
primary antibody (mouse-␣-TH; 1:500 dilution in PBS-TX
containing 0.1% BSA). The slices were rinsed three times
with PBS-TX-BSA and then incubated again at 37°C for 30
min with secondary antibody (goat-␣-mouse FITC, Sigma
Chemical, St. Louis, MO; 1:100 dilution in PBS-TX containing 0.1% BSA). The specimens were again rinsed three times
with PBS-TX-BSA solution and once with PBS-TX. Then the
specimens were mounted on glass slides and allowed to air
dry at room temperature, and mounted with one drop of
Fluoromount-G (Southern Biotechnology Associated, Birmingham, AL). Specimens were examined using a Nikon
E400 microscope fitted with epifluorescent filters for TRITC,
FITC, and ultraviolet light.
For TH and NOS dual immunochemical staining (group 5),
coverslips with adherent brain stem sections were preincubated at 37°C for 30 min with PBS-TX-BSA. The specimens
were rinsed once with fresh PBS-TX-BSA and incubated at
37°C for 2 h with a mixture of the primary antibodies [mouse␣-TH (1:500 dilution) and rabbit-␣-NOS (1:250 dilution) in
PBS-TX containing 0.1% BSA]. The specimens were rinsed
three times with PBS-TX-BSA, then incubated at 37°C for 30
min with a mixture of secondary antibodies [goat-␣-mouse
FITC (1:100 dilution) and goat-␣-rabbit TRITC (1:50 dilution) in PBS-TX containing 0.1% BSA]. The specimens were
rinsed three times with PBS-TX-BSA and once with PBS-TX.
The specimens were air dried at room temperature, mounted
on glass slides in Fluoromount-G, and examined with a
fluorescence microscope as described above.
The slices from rats in group 6 were rinsed with fresh
PBS-TX-BSA solution and incubated overnight at room temperature with a mixture of the primary antibodies [mouse-␣TH (1:500 dilution) and rabbit-␣-c-Fos (1:5,000 dilution) in
PBS-TX containing 0.1% BSA]. The slices were rinsed three
times with PBS-TX-BSA, then incubated at 37°C for 30 min
with a mixture of secondary antibodies [goat-␣-mouse FITC
(1:100 dilution) and goat-␣-rabbit TRITC (1:50 dilution) in
PBS-TX containing 0.1% BSA]. The specimens were again
rinsed three times with PBS-TX-BSA solution and once with
PBS-TX before being mounted on glass slides and air dried at
room temperature. The specimens were then mounted with
one drop of Fluoromount-G and examined with a fluorescence
microscope as described above.
Cells were counted on alternate brain stem slices by two
independent investigators who were unaware of the treatment. If the cell count differed by ⬎10%, a third investigator
then analyzed the brain stem slices. The final cell count was
the mathematical average of the independent cell counts.
To minimize errors in the counting of DMV somata, we
counted only those neurons in which the nucleus was clearly
visible. Despite this precaution, we have to consider cell
counts as best proportional estimates only, rather than absolute values, when comparing labeled subpopulations. This
is of paramount importance because the surface of the stomach wall covered by injection of rhodamine-coated beads was
limited to ⬃5 mm2. Therefore, this provided only a small,
though representative, fraction of labeled DMV neurons projecting to the gastric areas of interest.
The portion of the DMV located caudal to obex was defined
as the “caudal DMV” and the portion of DMV located rostral
to the anterior tip of the area postrema as the “rostral DMV.”
The area composed by the extension of the area postrema was
defined as the “intermediate DMV.” Cell count values are
given as means ⫾ SE.
Control experiments were carried out to ensure that the
antibody labeling was selective, namely: 1) incubation of
primary or secondary antibodies only and 2) reaction of
primary antibody with inappropriate secondary antibody. All
TH-IR IN RAT DMV
RESULTS
Sections of brain stem containing the DMV were
analyzed from ⬃2 mm caudal to the posterior tip of the
area postrema to ⬃3 mm rostral to the anterior tip of
the area postrema. The boundaries of the DMV were
delineated clearly by the presence of fluorogold-IR neurons (19, 24, 43). Only those neurons with a distinct
Fig. 1. Coronal view of the brain stem showing the distribution
within the dorsal motor nucleus of the vagus (DMV) of retrogradely
labeled neurons after microinjections of tetramethylrhodamine isothiocyanate (TRITC)-filled latex microspheres in the corpus, intraperitoneal injection of fluorogold to label DMV neurons, and immunocytochemical detection of tyrosine hydroxylase (TH). Computergenerated colors indicate the following: blue, fluorogold; green, TH
immunoreactive (TH-IR); red, TRITC microspheres; light blue, colocalization of fluorogold and TH-IR. A: caudal DMV. Note the heavy
density of TH-IR neurons within the DMV. Thick arrow, a corpusprojecting TH-positive neuron. B: intermediate DMV. Note the
larger number of corpus-projecting (rhodamine bead-filled) neurons,
1 of which contains TH-IR. C: rostral DMV. Note that because TH-IR
neurons lie outside the DMV, no corpus-projecting neuron contains
TH-IR. Thin arrows, fluorogold and TH-IR neurons. Thick arrows,
rhodamine-labeled and TH-IR DMV neurons. Thin convex arrows,
TH-IR neurons outside the DMV. Thick convex arrows, rhodaminelabeled DMV neurons (for clarity the rhodamine-labeled neurons in
B have not been indicated by arrows). CC, central canal; NTS,
nucleus of the solitary tract; XII, hypoglossal nucleus; IV, fourth
ventricle.
fluorogold-stained profile were counted. There were
998 ⫾ 62, 2,283 ⫾ 87, and 1,080 ⫾ 59 fluorogoldpositive neurons in the caudal, intermediate, and rostral DMV, respectively (n⫽ 6 rats).
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tests proved negative, indicating that the secondary antibodies were selective for their primary antibodies and that the
antibodies themselves exhibited neither nonspecific binding
nor excessive autofluorescence. Control experiments were
also carried out to ensure that there was no leakage or fading
of the fluorescence from the retrograde-labeled neurons due
to the immunochemical staining process. This was accomplished by comparison of the number of retrograde-labeled
neurons counted immediately after slicing and after the
immunochemical staining process.
The experiments were carried out in accordance with the
Henry Ford Health System guidelines for the care and use of
laboratory animals. All efforts were made to minimize pain,
reduce the number of animals used, and utilize alternatives
to in vivo techniques, if available.
Photographs were taken using a SPOT digital camera
mounted on a Nikon E400 fluorescent microscope. To reduce
the fluorescent light exposure time and the consequent fading of the fluorescence labeling, slices were photographed in
black and white using the appropriate filters, merged, and
assigned compute-generated colors (SPOT software, Diagnostic Instruments, Sterling Heights, MI).
Materials. TRITC latex microspheres (rhodamine beads)
were purchased from Lumafluor (Naples, FL). Nembutal was
purchased from Abbot Laboratories (North Chicago, IL). All
other chemicals were purchased from Sigma Chemical.
Composition of solutions. The fixative solution was composed of 32 g paraformaldehyde, 240 ml saturated picric acid,
5.25 g KH2PO4, 35.6 g Na2HPO4 䡠 7H2O, and 1,600 ml H2O.
The composition of the PBS solution was as follows: 13.5 g
NaCl, 40.2 g Na2HPO4 䡠 H2O, 2.04 g KH2PO4, and 1,500 ml
H2O. The PBS-TX solution was composed of 2.25 ml Triton
X-100 dissolved in 1,500 ml PBS buffer. The PBS-TX-BSA
solution composition was 1 g BSA dissolved in 100 ml
PBS-TX buffer.
Statistical analysis. Neuronal groups were compared using
the ␹2 test. The level of significance was set at P ⬍ 0.05.
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TH-IR IN RAT DMV
Fig. 2. Coronal view of the caudal
brain stem showing the distribution
within the DMV (outlined by the fluorogold-positive labeling) of TH and
nitric oxide synthase (NOS)-positive
neurons. Computer-generated colors
indicate the following: blue, fluorogold;
green, TH-IR; red, NOS-IR; light blue,
colocalization of fluorogold and TH-IR.
Note that the TH-positive DMV neurons (thick arrows) do not contain
NOS-IR (thin arrows). For clarity, only
a few selected DMV neurons have been
highlighted.
of the DMV colocalized with TH-IR (i.e., 13.7 ⫾ 1.1%;
n ⫽ 10 rats; P ⬍ 0.05 vs. fundus or antrum/pylorus).
Conversely, after injection of rhodamine beads in the
antrum/pylorus, 1 of the 41 rhodamine-labeled neurons in the caudal DMV also contained TH-IR (n ⫽ 8
rats); similarly after injection of rhodamine beads in
the fundus, 9 of the 216 rhodamine-labeled neurons
colocalized with TH-IR (n ⫽ 8 rats).
Figure 1 shows the distribution of dye-labeled neurons after injection of tracer in the gastric corpus,
intraperitoneal injection of fluorogold, and immunocytochemical detection of TH. As can be noted in the
micrograph (Fig. 1), most of the DMV neurons showing
colocalization of dye and TH-IR were located in the
caudal pole of the DMV whereas the majority of dyelabeled DMV neurons were in the intermediate portion
of the DMV. In Fig. 1, A and B (showing caudal and
intermediate brain stem section, respectively), DMV
neurons show colocalization of rhodamine beads and
TH-IR. In the rostral portion of the DMV (Fig. 1C),
TH-IR positive neurons are located outside the fluorogold-defined boundaries of the DMV.
Localization of TH- and NOS-positive neurons. In
the 10 brain stems analyzed, we never found any DMV
neurons that contained both NOS- and TH-IR (Fig. 2),
even in the caudal DMV, where both TH- and NOS-IR
positive neurons are densely located. In fact, despite
the observation that TH-IR and NOS-IR positive DMV
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Localization and projections of TH-IR neurons.
TH-IR neurons were distributed within the boundaries
of the DMV (as determined by fluorogold-IR) as well as
in the commissuralis, centralis, dorsalis, and medialis
subnuclei of the NTS. Focusing on the TH-IR neurons
in the DMV, the majority of the TH-IR neurons were
located in the caudal DMV where they comprised
10.9 ⫾ 0.6% of the total number of DMV neurons (i.e.,
108 ⫾ 12 TH-IR neurons of 998 ⫾ 62 fluorogold-positive neurons; n ⫽ 6 rats). Although there were more
fluorogold-labeled neurons in the intermediate portion
of the DMV, there were fewer TH-IR positive DMV
neurons present (i.e., 6 ⫾ 1 TH-IR neurons of 2,283 ⫾
86 fluorogold-positive neurons; n ⫽ 6 rats). In the
rostral portions of the DMV, the percentage of TH-IR
DMV neurons was 4.4 ⫾ 0.9% (i.e., 47 ⫾ 7 TH-IR
neurons of 1,080 ⫾ 58 fluorogold-positive neurons; n ⫽
6 rats). The total number of TH-IR neurons in the DMV
would then constitute a value similar to that previously found (22), though somewhat lower than in a
more recent study (39).
By applying retrograde-tracing techniques and immunochemical staining with specific antibodies against TH,
we analyzed the rostro-caudal span of the DMV for colocalization of rhodamine-labeled and TH-IR positive neurons, i.e., TH-IR neurons projecting to the gastric regions
of interest.
After injection of rhodamine in the corpus, 43 of the
368 rhodamine-labeled neurons in the caudal portion
TH-IR IN RAT DMV
DISCUSSION
The present study provides evidence that 1) TH-IR
positive neurons in the caudal DMV project selectively
to the gastric corpus; 2) the TH-IR positive DMV neurons comprise a neuronal subpopulation distinct from
the NOS-IR positive DMV neurons; and 3) TH-IR pos-
itive neurons in the DMV are not activated on esophageal distension.
Such evidence leads us to the following two conclusions. 1) The TH-IR positive neurons in caudal DMV
constitute a subpopulation of preganglionic neurons
distinct from the NOS-IR neurons. These TH-IR neurons do not seem to be implicated in the activation of
NANC inhibitory pathways of the gastric receptive
relaxation reflex activated by esophageal distension
(29, 43). 2) The discrete population of TH-IR positive
neurons in caudal DMV projecting to the corpus may
then comprise the preganglionic motoneurons involved
in gastric relaxation obtained via withdrawal of cholinergic tone (29, 34) or could constitute a subpopulation
of dopaminergic neurons involved in the attenuation of
stress or chemically induced ulcers (see Ref. 11 for
review).
The percentage of TH-IR positive neurons in the
caudal DMV reported in this study, i.e., 11% of the
fluorogold-labeled neurons, is similar to that found
previously by Manier et al. (22), though slightly lower
than in other reports (35, 39), supporting the validity of
our labeling techniques and cell-counting methods.
In this study, we report that ⬃14% of vagal neurons
projecting to the rat gastric corpus contain TH-IR. The
importance of such a relatively low percentage of
Fig. 3. Coronal view of the caudal
brain stem showing the distribution
within the DMV (outlined by the fluorogold positive labeling) of TH- and
c-Fos-IR positive neurons. Computer
generated colors indicate the following:
blue, fluorogold; green, TH-IR; red, cFos-IR; light blue, colocalization of fluorogold and TH-IR. Note that c-Fos
positive neurons (thin arrows) are either outside the DMV or inside the
DMV but do not contain TH (thin convex arrows). In fact, TH-IR neurons in
the DMV do not colocalize with c-Fos
(thick arrows).
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neurons were contained within the same region of the
caudal DMV, they were never colocalized.
Figure 2 is a micrograph showing the distribution of
TH- and NOS-IR positive neurons in the caudal portion of
the DMV. As can be noted, within the fluorogold-delimited boundaries of the DMV, the TH-IR positive neurons
are intermingled but not colocalized with the NOS-IR
positive neurons. The separation of TH- and NOS-IR
positive neurons is also present in the NTS area adjacent
to the DMV where the NOS-IR positive neurons are
located in patches dorsal to the TH-IR positive neurons.
Esophageal distension activates c-Fos expression. After esophageal distension for a period between 60 and
90 min (1 s on and 4 s off) we observed a consistent
pattern of c-Fos expression in the caudal dorsal brain
stem. At the level of the caudal DMV in all five brain
stems analyzed, although c-Fos-positive neurons were
present, we never found a DMV neuron that contained
both c-Fos- and TH-IR (Fig. 3).
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TH-IR IN RAT DMV
frequency coding (33, 42), suggests that the vagus
nerve may encode the release of different neurotransmitters according to the firing frequency at which it is
driven. Indeed, a frequency-response code seems to
exist in both the NTS after esophageal distension (see
Fig. 2 in Ref. 29) and the DMV after injection of direct
current (5). It is then possible that the frequency code
necessary to activate TH-IR positive neurons in DMV
is not the same as that activated by our esophageal
distension paradigm.
Second, though less probable, is that TH-IR neurons
in DMV are not involved in gastric distension reflexes
but rather in the mucosa protection exerted by dopamine (11). Such a scenario is unlikely since previously
discussed evidence (27, 37) provides a rather strong
case in favor of involvement of DMV catecholaminergic
neurons in gastric relaxation.
In conclusion, we have shown that a significant percentage of caudal DMV neurons projecting to the stomach corpus contain TH-IR and are not colocalized with
NOS-IR positive neurons. In addition, we suggest that
the TH-IR positive neurons may be involved in the
withdrawal of cholinergic tone to the gastric corpus.
This work was supported by National Science Foundation Grant
9816662 (R. A. Travagli) and National Institute of Diabetes and
Digestive and Kidney Diseases Grant DK-56373 (R. Rogers).
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TH-IR neurons in the DMV should not be underestimated, because single vagal motoneurons project extensively to adjacent areas of the gastrointestinal tract
(14) and could thus influence selectively similar functions.
Our data showing lack of colocalization of TH- and
NOS-IR in caudal DMV neurons extend a previous
observation by Ohta and colleagues (23). In that study
(23), evidence was provided that NOS neurons did not
colocalize with catecholaminergic neurons in the adjacent medial NTS subnucleus.
Using both in vitro and in vivo techniques, several
groups have shown recently that a vast array of electrical (36, 38), physiological (27, 29), or mechanical (8,
25, 37) stimuli can activate DMV motoneurons and
induce gastric relaxation (8, 27, 29). Some of these
stimuli, i.e., peripheral injection of CCK (27) or gastric
(37) or esophageal distension (29), selectively activate
the caudal DMV neuronal population.
Vagally mediated cholinergic stimuli increase gastric functional parameters, including tone and motility
(10). In several studies (9, 30, 31, 41, 42), it has been
demonstrated that activation of presynaptic ␣2-adrenoceptors decreases vagally evoked ACh release to the
stomach and that exogenous application of norepinephrine decreases ACh release from vagal terminals in
both the corpal (30) and antral portions of the stomach
(32). In addition, the decrease in gastric motility that
follows peripheral CCK administration involves the
activation of a vagal-dependent mechanism (12, 26,
27). It is established that both endogenous and exogenous CCK stimulate distension-sensitive vagal afferents, producing distension-like effects on vagal neurons (26) and expression of c-FOS in TH-IR positive
DMV neurons (27).
Using an esophageal balloon distension protocol
proven to induce gastric relaxation (29), we were unable to activate c-Fos expression in TH-IR positive
caudal DMV neurons. Our data thus suggest that the
origin of the adrenergic modulation of ACh release
from vagal terminals must be found in areas other
than the caudal DMV.
Because our esophageal stimulation paradigm did
not activate TH-IR caudal DMV neurons (otherwise we
would have observed an increase in c-Fos activity), the
following two scenarios may be considered.
First, TH-IR positive neurons in the caudal DMV
may be implicated in withdrawal of cholinergic tone by
an activation of GABA terminals via a mechanism
unrelated to esophageal distension. In fact, adrenergic
blockade with ␣- or ␤-adrenoceptor antagonists did not
seem to affect the relaxatory or contractile responses
elicited by either electrical vagal stimulation or by
esophageal or duodenal distension (7, 33). At the same
time, however, nicotine perfusion, in the presence of
the muscarinic receptor antagonist atropine, induced a
relaxation of the gastric fundus that was mediated
largely by NO release and partially by the release of
norepinephrine (16). The observation that vagal inhibitory and excitatory fibers are not simultaneously activated by gastric distension (34), but rather require a
TH-IR IN RAT DMV
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